The genomes of many positive sense RNA viruses contain an internal ribosomal entry site (IRES) that mediates end-independent initiation of translation. IRESs belong to different structural classes and use distinct mechanisms for initiation. Our proposed studies of the process of initiation on representatives of four classes of IRES that use mechanisms of increasing complexity will identify important cis-acting elements and provide detailed mechanistic insights into the actions of canonical initiation factors and cellular IRES trans-acting factors (ITAFs) in promoting internal ribosomal entry. Our studies will address mechanisms of increasing complexity. The ~180nt-long intergenomic region (IGR) IRESs of dicistroviruses mediate initiation without initiator tRNA or initiation factors and the first elongation cycle consequently occurs without deacylated tRNA in the ribosomal P site. We will characterize whether elements within the IRES play an analogous role during elongation to deacylated tRNA when it has been translocated to the E site. The IRES of the pestivirus classical swine fever virus is substantially resistant to inhibition by eIF2 phosphorylation, which can be accounted for by the use of two exceptional eIF2- independent mechanisms of initiation. We will characterize whether these mechanisms account for relaxation on this IRES of the ribosome[unreadable]s normally strict avoidance of initiation at non-AUG codons. Type 2 picornavirus IRESs such the encephalomyocarditis virus IRES bind specifically to eIF4G/eIF4A: we will investigate how these factors act to promote recruitment of 43S complexes to the initiation codon, how ITAFs promote binding of eIF4G/eIF4A and why only a subset of type IRESs require their activity. The mechanism of initiation on type 1 picornavirus IRESs, such as that of poliovirus, is not known. We shall characterize it by purifying and identifying all necessary factors in order to reconstitute the entire initiation process in vitro. We will then characterize interactions between factors and the ribosome using chemical/enzymatic footprinting and directed hydroxyl radical cleavage. The studies will provide a framework for understanding of mechanistic details of IRES-mediated initiation, for understanding the cell-type specificity of IRES function and for the design of inhibitors.